南开大学 基因操作原理-第十二章

GENETRANSFERTOPLANTSCHAPTER12YingWang,Ph.D.,TEDASchoolofBiologicalSciencesandBiotechnologyNankaiUniversityyingwang@1GENEMANIPULATIONINPLANTSAdvantages:

Totipotencyandregeneration Varietiesandmutants

Progeny SelfedDisadvantages:Quantitativetrait Genomesize Lifecycle Multiploid21.

Candidategenes2.Plantmaterial3.Transformationmethods

Agrobacterium-mediated Plantvirus-mediated DirectDNAtransfer4.SelectionandregenerationOVERVIEW3CANDIDATEGENES(I)Selectablemarkergenes:

neomycinphosphotransferaseIIgene,nptIIhygromycinphosphotrasferasegene,hptchloramphenicolacetyltransferase(CAT)gene,

cat4CANDIDATEGENES(II)Reportergenes:b-glucuronidase(GUS)gene,gusLuciferase(LUC)gene,lucGreenfluorescentprotein(GFP)gene,gfp5CANDIDATEGENES(III)Insect-resistantgene:

cryHerbicide-resistantgene:

barVirus-resistantgene:

cp,polBacterial-resistantgene:

lyticpeptidegene,lysozymegeneStress-resistantgene:6PLANTMATERIALCallus:Suspensionculturecell:Protoplast:Explantorplanta:7CALLUSEquimolaramountsofauxinandcytokininstimulatecelldivision.Leadstoaproliferationofanundifferentiatedmassofcellscalledacallus.Avilés,F.,etal.,(2009)EffectofCultureMediuminCallogenesisfromAdultWalnutLeaves(JuglansregiaL.)ChileanJournalofAgriculturalResearch,69(3):460-467.81.Desirablepropertiesofexplant: Easilysterilisable Richinundeterminedcells Responsibletoculture2.Oftenusedexplant: Shoot/roottips Axillarybuds Hypocotyl LeavesCALLUS-INDUCEDEXPLANT9WaterInorganicsaltformulations

macro-elements(>mg/l) traceelements(<mg/l).Sourceofcarbohydrate

usuallyaddedassucroseat2-3%(w/v)Vitaminsandorganicsupplements

nicotinicacid,thiamine,pyridoxine,glycine,inositolPlanthormonesandgrowthregulators

Solidifyingagents

UndefinedsupplementsCALLUSCULTUREMEDIUM10PLANTHORMONESAbsolutelyessentialPromoterootingAbsolutelyessentialInduceshootsformationPromotecellandstemelongation,breakdormancyPromotesleafabscissionandseeddormancyInvolvedinwoundresponsesIndole-3-aceticacid(IAA)[anauxin]Zeatin[acytokinin]EthyleneGibberellicacid(GA3)[agibberellin]Abascisicacid(ABA)11Cytokinin(shoot)Auxin(root)LeafstripShootRootCallusAUXINvs.CYTOKININ12CELL-SUSPENSIONCULTURE1.Whencallusisagitatedinaliquidmedium,thecellmassbreaksup,formingcellsuspension.2.“Friable”callusgoeseasilyintosuspension:2,4-D;Lowcytokinin;semi-solidmedium; pectinasedigestion;blending

3.Extremelyheterogeneous,includingisolatedcells,smallclustersofcellsandmuchlargeraggregates.4.Canbemaintainedindefinitely,somaclonalvariation5.Noneedforsolidsupport,easytobescaled-up13PROTOPLAST1.Protoplastsarecellsfromwhichthecellulosewallshavebeenremoved.2.Usefulmaterialforgeneticmanipulationinplants.3.MechanicaldisruptionPectinase&cellulasetreatmentProtoplastSuspensioncultures,callusorintacttissuesCentrifugation14Interconversion&Regeneration15Vector-mediatedgenetransfer Agrobacterium-mediated Plantvirus-mediatedDirectDNAtransferPLANTTRANSFORMATON16CROWN-GALLDISEASE17CROWN-GALLDISEASE1.AplanttumorinducedbysoilbacteriumAgrobacterium2.Dicotswithwoundsites3.Trueoncogenetransformation:

unlimitedgrowthinabsenceofphytohormones calluscanbecultivatedinvitroevenifthebacteriaarekilled4.SynthesisofopinesAnaturalplantgeneticengineering181.AgrobacteriumtumefaciensGramnegativebacteria192.Signalmoleculesproducedbywoundedplants:203.Trueoncogenetransformation:WoundinfectedwithA.tumefaciensCrowngallExcisetissuefromcrowngallTissuegrowsascallusintheabsenceofphytohormoneAddcarbenicillintokillAgrobacterium214.OPINES:unusualaminoacidderivativesSynthesizedbyplantcellsDeterminedbyAgrobacteriumtumefaciensUsedassolecarbonandnitrogensourceOctopinefamilyNopalinefamily22Tumor-inducing(Ti)plasmidZaenen,I.,VanLarbek,N.,Teuchy,H.,VanMontagu,M.andSchell,J.,Super-coiledcircularDHAincrown-gallinducingAgrobacteriumstrains.J.Mol.Biol.86:109-127,1974Super-coiledcircularDNAwith140-235kb23T-DNAtms:auxin(shootytumor)tmr:

cytokinin(rootytumor)nos:

nopalinesynthaseocs:

octopinesynthaseTLsegment:genesfortumorformationTRsegment:genesforopinesynthesisborder:25bpinperfectdirectrepeats24VirulencegenesvirAandvirGareconstitutivelyexpressed;virB,

virC,virDandvirEareinduced.VirA:akinasespanningtheinnerbacterialmembraneandactasareceptorforwoundsignal.ActivatedVirAtransphosphorylatestheVirGprotein.VirG:aDNA-bindingproteinandatranscriptionalactivatorofothervirgenes.VirB:formscomponentsoftheconjugativepilusthroughwhichtheT-DNAistransferredtoplantcells.VirD:endonucleasewhichintroduceseitherss-nicksords-breakatthe25bpborders.VirC:enhanceroftheendonucleasereactioncatalyzedbyVirD.VirE:single-strandedDNAbindingprotein,protectingT-DNAfromdegradation.25Tzfira,T.,et.al.(2004)AgrobacteriumT-DNAintegration:moleculesandmodels.Trends.Gen.,20(8):375-383.26AgrobacteriumtransferT-DNAintoplantchromosome27Tzfira,T.andCitovsky,V.(2006)Agrobacterium-mediatedgenetictransformationofplants:biologyandbiotechnologyCurr.Opin.Biotechnol.17:147-15428DISARMEDTi-VECTORForselectionDisarmedvectors:donotproducetumors;canbeusedtoregeneratenormalplantscontainingtheforeigngene.29disarmedTi-vectorIntermediatevectorHomologousrecombinationbetweenpBR322DNAregionsKanamycinselectionCOINTEGRATEVECTOR30TRIPARENTALMATINGTriparent:1.E.colistraincarryingahelperplasmidabletomobilizetheintermediatevector2.E.colistraincarryingtherecombinantintermediatevector(IV)3.A.tumefacienscarryingthedisarmedTiplasmidE.coliIVcolE1oribom=basisofmobilizationE.colihelperoribompGV3850disarmedTiplasmidA.tumefaciensTioriV31STEPIHelperplasmidmobilizesitselfinto2nd

E.colistraincontainingintermediatevector.E.coliIVcolE1oriE.colihelperoribom+E.colihelperIV32STEPIIHelperplasmidmobilizesitselfandtheintermediateplasmidintoAgrobacterium.E.colihelperIVpGV3850disarmedTiplasmidA.tumefaciensTioriV+cannotreplicateA.tumefacienspGV3850disarmedTiplasmidTioriVhelperIV33BINARYVECTORSYSTEM

modifiedTiplasmidoriVvirT-DNAdeleted2LBRBorikanrpolylinker134BINARYVECTOR35LEAFDISC36DICOTSvs.MONOCOTS1.Woundedmonocottissuesdonotproducephenolics;

pretreatwithwoundexudatefromdicots2.TiVirulence

IncreasevirulencebyincreasingtheexpressionofvirGandvirE13.Woundsitesinmonocotstendtobecomelignified;

useexplantscontaininghighproportionofactivelydividingcells4.nptIIgenecannotbeusedasselectablemarker

usehptgene(hygromycinR)5.Riplasmid37PLANTVIRUSVECTOR1.Plantvirusesareabletoabsorbtoandintroducetheirnucleicacidsintotheplantcells；2.Infectedcellsyieldlargeamountsofvirus,sorecombinantviralvectorshavethepotentialforhighleveltransgeneexpression；3.Viralinfectionsareoftensystemic,consistent,rapid；4.plantvirusesneitherintegrateintonorpassthroughthegermline.Caulimoviruses---dsDNA–CaMVGeminiviru